ABSTRACT This R21 is focused on the discovery of fundamental knowledge about the nature and behavior of living systems. One of the fundamental challenges in developmental biology is delineating hierarchical cellular states including rare intermediates and the corresponding networks of regulatory genes that mediate cell-type specification. Single-cell RNA-Seq (scRNA-Seq) provides comprehensive descriptions of genomic states including those of rare intermediates and their presumptive regulators. We recently utilized scRNA-Seq and a new bioinformatics tool called Iterative Clustering and Guide Gene Selection (ICGS), as well as clonogenic assays to delineate hierarchical genomic and regulatory states culminating in neutrophil or macrophage specification. Strikingly, we found that myeloid cells undergoing specification traverse successive states of counter-acting transcriptional programs, which appear to be obligatory during cell-fate determination. We propose to extend this work by elucidating the key mixed-lineage intermediates that are traversed during the specification of innate and adaptive lymphocyte precursors. We will use diverse flow cytometric sorting strategies to capture a continuum of developmental intermediates undergoing innate and adaptive immune cell specification. Captured cells will be used to generate deep scRNA-Seq libraries, followed by ICGS analysis. Next, we will exploit a new lineage tracking tool to rigorously map the developmental trajectories of progenitors as they traverse via metastable intermediates to specified states in an unperturbed hematopoietic system. The proposed work will identify key mixed-lineage states that are traversed during the specification of innate and adaptive lymphocyte precursors. As such it will clarify the developmental origins of innate and adaptive lymphocytes in the hematopoietic system. Furthermore such analyses will set the stage for revealing new regulatory interactions between known transcription factors as well as new transcription factors that dictate development of innate and adaptive immune cells.